Occurrence, isolation, and characterization of polyribosomes in yeast.

This report details the procedural requirements for preparing cell-free extracts of yeast rich in polyribosomes. This enabled us to demonstrate the occurrence of polyribosomes in yeast, to show their role in protein synthesis, and to devise methods for their resolution and isolation. When certain precautions are met (the use of log phase cells, rapidly halting cell growth, gentle methods of disruption, sedimentation through exponential density gradients, etc.), individual polyribosome size classes ranging up to the heptosome can be fractionated and separated from their nearest neighbors. Larger size classes are resolved partially among themselves, free of smaller polyribosomes. This was confirmed by extensive electron micrographic studies of material from the various fractions obtained upon density gradient centrifugation of yeast extracts. Modifications of the gradients and procedure should allow fractionation and isolation of the larger polyribosomes, including those containing polycistronic messages. Yeast polyribosomes are disaggregated to single ribosomes by longer term grinding, cell disruption by the French pressure cell, the Hughes press, or by incubation with dilute RNAse. Yeast polyribosomes are active in the incorporation of amino acids into polypeptide; the single ribosomes exhibit only slight activity. The latter activity is probably due to the presence of a small fraction of monosomes still containing mRNA. Poly-U stimulates amino acid incorporation only in the single ribosomes.

The biogenesis of mitochondria. 3. The lipid composition of aerobically and anaerobically grown Saccharomyces cerevisiae as related to the membrane systems of the cells.

The growth conditions known to influence the occurrence of mitochondrial profiles and other cell membrane systems in anaerobic cells of S. cerevisiae have been examined, and the effect of the several growth media on the lipid composition of the organism has been determined. The anaerobic cell type containing neither detectable mitochondrial profiles nor the large cell vacuole may be obtained by the culture of the organism on growth-limiting levels of the lipids, ergosterol, and unsaturated fatty acids. Under these conditions, the organism has a high content of short-chain saturated fatty acids (10:0, 12:0), phosphatidyl choline, and squalene, compared with aerobically grown cells, and it is especially low in phosphatidyl ethanolamine and the glycerol phosphatides (phosphatidyl glycerol + cardiolipin). The high levels of unsaturated fatty acids normally found in the phospholipids of the aerobic cells are largely replaced by the short-chain saturated acids, even though the phospholipid fraction contains virtually all of the small amounts of unsaturated fatty acid present in the anaerobic cells. Such anaerobic cells may contain as little as 0.12 mg of ergosterol per g dry weight of cells while the aerobic cells contain about 6 mg of ergosterol per g dry weight. Anaerobic cell types containing mitochondrial profiles can be obtained by the culture of the organism in the presence of excess quantities of ergosterol and unsaturated fatty acids. Such cells have increased levels of total phospholipid, ergosterol, and unsaturated fatty acids, although these compounds do not reach the levels found in aerobic cells. The level of ergosterol in anaerobic cells is markedly influenced by the nature of the carbohydrate in the medium; those cells grown on galactose media supplemented with ergosterol and unsaturated fatty acids have well defined mitochondrial profiles and an ergosterol content (2 mg per g dry weight of cells) three times that of equivalent glucose-grown cells which have poorly defined organelle profiles. Anaerobic cells which are low in ergosterol synthesize increased amounts of squalene.

Cardiolipin content of wild type and mutant yeasts in relation to mitochondrial function and development.

The phospholipid composition of various strains of the yeast, Saccharomyces cerevisiae, and several of their derived mitochondrial mutants grown under conditions designed to induce variations in the complement of mitochondrial membranes has been examined. Wild type and petite (cytoplasmic respiratory deficient) yeasts were fractionated into various subcellular fractions, which were monitored by electron microscopy and analyzed for cytochrome oxidase (in wild type) and phospholipid composition. 90% or more of the phospholipid, cardiolipin was found in the mitochondrial membranes of wild type and petite yeast. Cardiolipin content differed markedly under various growth conditions. Stationary yeast grown in glucose had better developed mitochondria and more cardiolipin than repressed log phase yeast. Aerobic yeast contained more cardiolipin than anaerobic yeast. Respiration-deficient cytoplasmic mitochondrial mutants, both suppressive and neutral, contained less cardiolipin than corresponding wild types. A chromosomal mutant lacking respiratory function had normal cardiolipin content. Log phase cells grown in galactose and lactate, which do not readily repress the development of mitochondrial membranes, contained as much cardiolipin as stationary phase cells grown in glucose. Cytoplasmic mitochondrial mutants respond to changes in the glucose concentration of the growth medium by variations in their cardiolipin content in the same way as wild type yeast does under similar growth conditions. It is concluded that cardiolipin content of yeast is correlated with, and is a good indicator of, the state of development of mitochondrial membrane.

Separation of spores from diploid cells of yeast by stable-flow free-boundary electrophoresis.

By the use of stable-flow free-boundary (Staflo) electrophoresis and the electrophoretic mobility difference between ascospores and diploid cells of Saccharomyces cerevisiae, a mixture of the two can be separated into spore and diploid cell fractions. The spore fraction that is obtained can then be used for genetic analysis.

Cytokinin activity: localization in transfer RNA preparations.

Transfer RNA from yeast, liver, and Escherichia coli has cytokinin activity in the tobacco callus bioassay, whereas ribosomal RNA from yeast is inactive. In contrast to fractions of yeast transfer RNA rich in serine acceptor and cytokinin activity, preparations (70 to 90 percent pure) of arginine transfer RNA(2), glycine transfer RNA, phenylalanine transfer RNA, and valine transfer RNA(1) and of highly purified alanine transfer RNA from yeast were inactive at concentrations of 20 to 2500 micrograms per liter. One molecule of 6-(gamma,gamma-dimethylallylamino) purine per 20 molecules of yeast tRNA would account for the observed cytokinin activity. The number of major molecular species contributing to cytokinin activity of transfer RNA, therefore, must be small.

Localization of polyphosphates at the outside of the yeast cell plasma membrane.

Under appropriate experimental conditions toluidine blue is bound to the yeast cell surface, without penetrating into the cells. Based on experimental observations it is highly probable that the dye is bound to polyphosphates, localized outside the plasma membrane. The probable localization of polyphosphates outside the plasma membrane is important in the context of the proposed involvement of polyphosphates in glucose transport in yeast.

Levels and turnover of the proteinase B inhibitors in yeast.

The ratio of the proteinase B inhibitors IB1 and IB2 from baker's yeast was shown to depend on the yeast strain by specific immunoprecipitation from boiled yeast extract and subsequent electrophoresis of the heat-dissociated precipitates on polyacrylamide gels. Bothe IB1 and IB2 were found, IB2 being by far predominant. Saccharomyces carlsbergensis NCYC 74 contained IB1, whereas in Saccharomyces cerevisiae X 2180 only IB2 was present. When cells of the latter strain were labelled with [14C] leucine from the beginning of growth and pulsed with [3H] leucine during the stationary phase, no short-lived IB1 could be detected. However, the peak of IB2 resolved on the gel showed an increased 3H/14C ratio in comparison to the majority of the other cellular proteins. The increased 3H/14C ratio was found to be the result of catabolite repression of inhibitor synthesis during exponential growth: cells growing on glucose as carbon source contain high inhibitor levels only during the stationary phase of growth, whereas during growth on acetate high amounts of inhibitor are present even in exponentially growing cells. During the stationary phase of growth the inhibitor is degraded with the same half-life as the total cellular proteins (about 50 h).

The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane.

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (gz) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. The tetranuclear iron-sulfur clusters can have their gz axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.

Sequence homology of nuclear and mitochondrial DNAs of different yeasts.

1. Both nuclear and mtDNA of four different yeasts show approximately 10% homology as measured by DNA-DNA filter hybridization. These homologous sequences are mainly attributable to the ribosomal cistrons. 2. Melting curve analysis shows that the heterologous mitochondrial DNA-DNA hybrids contain several times more mismatching than the nuclear DNA-DNA hybrids. 3. DNA-rRNA hybridization shows that the sequences of the ribosomal cistrons in both the nuclear and the mitochondrial genome have been conserved during evolution. 4. However, melting curve analysis of the DNA-RNA hybrids shows that the sequence of the nuclear ribosomal cistrons have undergone considerable fewer nucleotide substitutions than their mitochondrial counterparts. 5. The results suggest that the mitochondrial ribosomal cistrons have evolved more rapidly than the nuclear cistrons. This is discussed in the light of theories on the rat of molecular evolutin.

Electrophoretic behaviour of yeast mitochondrial translation products.

We have studied the mobility of yeast mitochondrial translation products during electrophoresis on polyacrylamide gels of different composition and found that these polypeptides can be divided into two groups. One, to which subunit II of cytochrome c oxidase belongs, behaves normal as all water-soluble reference proteins. The other, to which cytochrome b and subunits I and III of cytochrome c oxidase belong, shows a free electrophoretic mobility about twice as fast as the first group. Conditions have been found to separate cytochrome c1 from cytochrome b.

Identification of yeast 60 S ribosomal proteins crosslinked to rRNA by 2-iminothiolane.

Saccharomyces carlsbergensis 60 S ribosomal subunits were treated with the hetero-bifunctional crosslinking agent 2-iminothiolane and then subjected to mild UV irradiation to introduce protein-rRNA crosslinks. The major crosslinked products were identified as proteins L2, L3, L5, L19 and L23 of which L5 was found to be crosslinked at a 3-5-fold higher efficiency than the other four. Several additional proteins were cross-linked to a detectable but much lower extent.

[Characterization of cell wall constituents involved in the flocculation of Saccharomyces uvarum yeast strains]. / Caractérisation des constituants pariétaux impliqués dans la floculation de levures Saccharomyces uvarum.

Cell walls of flocculent strains (0006) and non flocculent strains (0019) of Saccharomyces uvarum (Carlsbergensis), grown in different media and taken in both growth and stationary phases, were treated with water and with 2 per cent (W/V) potassium hydroxide. This treatment yielded four fractions (FI, FII, FIII and FR). The fractions FI isolated from the flocculent cell walls contained more mannose and less protein than the corresponding fractions FI isolated from the non flocculent cell walls. The amino-acid composition was also different between the two types of fractions. A radioactive labelling technique revealed that the FI and the walls from flocculent cells bound on average two to three times as much 45Ca as did the FI and the walls from non flocculent yeast. The substitution of carboxyl groups in FI and walls with glycine methyl ester led to a great drop of the 45Ca binding capacity. This result suggests that carboxyl groups of the cell walls are involved in the flocculation process. But flocculation seems to be a phenomenon more complex than the simple formation of a Ca2+ bridge, the involvement of "lectin like" components easily removed from the cell walls, should not be rejected.

Peptide elongation factor 1 from yeasts: purification and biochemical characterization of peptide elongation factors 1 alpha and 1 beta (gamma) from Saccharomyces carlsbergensis and Schizosaccharomyces pombe.

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) [corrected] was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S. carls.) and Schizosaccharomyces pombe (S. pombe). The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts. The basic proteins have a molecular weight of 47,000 for the S. carls. factor and of 49,000 for the S. pombe factor. While the purified yeast EF-1 alpha s function analogously to other eukaryotic factors and the E. coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of Phe-tRNA in the absence of poly(U) as mRNA. This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s). Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively. Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 alpha such as GDP, GTP, and aminoacyl-tRNA. Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S. carls. EF-1 alpha was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S. cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S. cerev. sequence at the protein level. EF-1 beta and EF-1 beta gamma were isolated and highly purified as biologically active entities from the two yeasts. EF-1 beta s from the two yeasts have the same molecular weight of 27,000, whereas component gamma of the S. carls. EF-1 beta gamma showed a higher molecular weight (47,000) than that of the S. pombe factor (40,000). It was also shown that a stoichiometric complex was formed between EF-1 alpha and EF-1 beta gamma from S. pombe. Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-1 alpha beta gamma) fraction from freshly prepared cell-free extracts of yeast.

Neutral lipids in the cells and cell envelope fractions of aerobic baker's yeast and anaerobic brewer's yeast.

The neutral lipids from whole cells and cell envelopes of aerobic Saccharomyces cerevisiae and anaerobic Sacch. carlsbergensis and the cell walls isolated from the cell envelopes were analysed. The effect of anaerobiosis was particularly clear on the neutral lipid composition of the plasma membrane. Compared to the anaerobic membrane, the aerobic membrane contained more C16:1, C18:1 and other unsaturated fatty acids, more total sterol, more than ten times as much ergosterol and less than one tenth as much squalene, reflecting differences between the aerobic and anaerobic whole cellmthe main sterol in the aerobic membrane ergosterol, was mainly in the free form, whereas zymosterol, 24(28)-dehydroergosterol, epi- or fecosterol and lanosterol were predominantly esterified. In contrast, the anerobic membrane contained small amounts of biosynthetic sterol precursors of ergosterol (mainly esterified), and was clearly richer in saturated fatty acids having a greater variation in chain length and in 18:2 acid. Both plasma membranes contained a considerable amount of triacyglycerols, while the amount of lower acylglycerols was clearly higher in the anaerobic plasma membrane. The lipid composition of both cell walls were relatively similar, consisting mainly of triacylglycerols and lower acylglycerols.

Plasma vitamin B6 concentrations in Nigerian adolescents.

Vitamin B6 status was assessed from dietary and plasma vitamin B6 concentration using Saccharomyces uvarum as test organism, and erythrocyte alanine amino transferase activity (E-ALAT). The subjects participating in the study were 72 males and 30 females (aged 10-18 years) who resided in a boarding institution. Mean daily dietary vitamin B6 and protein intakes were 1.56 +/- 0.42 mg and 63.0 +/- 9.6 g respectively. The corresponding mean plasma vitamin B6 concentration was 194 +/- 44.2 nmol/l. Neither age, sex nor menarche had significant effect (P less than 0.05) on plasma vitamin B6 concentration of these adolescents. Dietary vitamin B6 but not protein intake correlated with plasma vitamin B6 (r = 0.3076, P less than 0.002). However, low dietary vitamin B6/protein ratio (less than 0.02 mg/g) was not reflected in plasma vitamin B6 concentration, but low plasma vitamin B6 concentration (120-179 nmol/l) corresponded to low E-ALAT activity after in vitro addition of pyridoxal phosphate (E-ELAT 16 per cent). A stimulation above 25 per cent, 16-25 per cent and below 16 per cent was used as an indicator of poor, marginal and adequate vitamin B6 status, respectively. Based on these criteria 30.7 per cent, 17.8 per cent and 51.5 per cent of subjects, with corresponding mean plasma vitamin B6 of 150 +/- 28.4, 192 +/- 8.5 and 237 +/- 18.7 nmol/l are of deficient, marginal and adequate vitamin B6 status, respectively.

["Killer" plasmid in natural strains of Saccharomyces]. / Plazmida "killer" u prirodnykh shtammov Saccharomyces.

Double stranded RNA's of thirteen wine strains of Saccharomyces belonging to different species and exhibiting killing activity were studied. Two strains of Oxford genetic stocks were used for control. All these strains have four different spectra of killer activity and contain two double stranded RNAs, L and M. All strains regardless of the type of killer activity have the same electrophoretic mobility of L. The mobility of M varies in different strains but is not connected with the spectrum of killing activity. All strains can be cured by cycloheximide. Cured variants have no M. Certain cured strains contain small portions of double stranded RNAs with electrophoretical mobility faster than of M.